Profiling urinary steroids. A reliable procedure

CLINICA CHIMICA ACTA CCA 4770

PROFILING

URINARY

J. F. VAN DE CALSEYDE**,

STEROIDS.

A RELIABLE

R. J. H. SCHOLTIS,

PROCEDURE*

N. A. SCHMIDT

AND C. J. J. A. LEIJTEN

Department of Clinical Chemistry, Ignatius Hospital, Breda (The Netherlands) (Received

September

7, 1971)

SUMMARY

A relatively simple method of gas chromatography, permiting the quantitative estimation of the most important urinary r7-ketosteroids and 17-OH corticosteroids, is reported. The procedure consists in enzyme hydrolysis, dichloromethane extraction, and (without any further purification) gas-liquid chromatography of the steroids as their o-methyloxime-trimethyl silyl ether (MO-TMS) derivatives. The method is shown to be useful in clinical studies.

INTRODUCTION

An increasing number of papers dealing with the analysis of urinary steroids are concerned with methods designed to estimate the majority of urinary metabolites important for endocrinological diagnosis, by a single chromatographic procedure. Gas-liquid chromatography (GLC) IS . one of the most widely used procedures, although promising results have also been obtained using a modern method of liquid-liquid chromatographyl. Until recently, profiling steroids by GLC was confined to r7-ketosteroids, pregnanediol and pregnanetrio12. Analysis of cortisol-like steroids was hindered by the thermal instability of these compounds3 and the fact that they could not be protected by the production of derivatives because of steric hindrance. Excellent work by Horning and his co-workers4~5 showed, however, that these steroids may be determined by gas chromatography using MO-TMS derivatives. The present studies were designed to develop a procedure which, in conjunction with proved methods, would make it possible for the clinical chemist to contribute towards endocrinological diagnosis. Therefore, methods not yet sufficiently tested in practice, such as capillary columns and mass spectrometry677, were not used to refine the present procedure qualitatively or quantitatively. Evaluation of packed capillary columns will possibly offer a genuine prospects. * Requests for reprints to R. J. H. Scholtis, Ignatius Hospital, Breda, the Netherlands. ** Present address: St. Canisius Hospital, Nijmegen, the Netherlands. Abbreviations : BSA = N, 0-bis-(trymethylsilyl) TSIM = trimethylsilylimidazole,

acetamide,

TMCS =

trimethylchlorosilane,

C&z. Chim. Acta, 38 (1972) 103-111

104

VAN

MATERIAL

AND

DE CALSEYDE

et d.

METHODS

Sf!wcinten One ml of 3 M acetate buffer, pH 4.6, containing approximately 50 mg of boric acid, was added to IOO ml of a 24-h urine sample. The urine was saturated with chloroform and centrifuged

before analysis.

Urines stored for subsequent

examination

were

frozen9.

0.6 ml of I M acetate buffer, pH 4.6 and 0.35 ml of Helix pontatia” juice were added to 6 ml of urine. The mixture was stored in the incubator at 50~ for 20 h.

After cooling and adding 2.5 g with IO ml of dichloromethane. The of tetracosan as an internal standard. of a NaOH-carbonate solution (0.5

of sodium chloride, the urine was extracted twice first portion of dichloromethane contained 20 ,ug The pooled dichloromethane was washed in I ml M NaOH and I M Na,CO,) and subsequently in

0.5 ml of I M acetate buffer, pH 4.6. The dichloromethane was dried with approximately 2.5 g of anhydrous Na,SO, and then evaporated at 4.5’.

The methoxime derivatives were prepared as follows: the residue was dissolved hydrochloride solution in pyridine. The solution was in 100$ of a r.5y0 methosamine allowed to stand 90 min at 90~. The mixture was evaporated to dryness with nitrogen and silylation was carried out with 60 ~1 of a silylation mixture (BSA-TSI~~-TALCSpyridine, 9: 9: 6: I, for at least 5 h at Go”. Of this solution, 2 ~1 were injected into the gas chromatograph. Gas clironiatografdi~~ A Becker 420 gas chromatograph

was used, which was fitted with FID and glass

columns measuring 2 mx2.6 mm (i.d.). Stationary phase: 30/b OV-I on Gas-Chrom Q 100-120 mesh (Applied Science). Temperatures: programming from 190’~250°, z”C/min. Inlet and detector 260’. Carrier gas: nitrogen, 45 ml/min.

Starting from 6 ml of distilled water in lieu of urine, operations similar to those adopted in the assay were carried out. The initial extraction was performed with dichloromethane containing 20 pg of internal standard (IS) and zo pg of the following steroids: A, E, D, KE, HE, Pz, P,, THS, THE, and THF (for abbreviations, see Table I). From the chromatogram produced, a factor was calculated for each of the steroids : surface

area of IS

’ = KZZ&rZof

* Inclustric

IhAogique

Steroid

Franpise.

PROFILING

URINARY

105

STEROIDS

The steroid concentrations

in the unknown urine then were calculated as follows:

surface area of steroid G&face area of IS

x f x T = mg of steroid/litre.

Quality control (I) Possible

losses of fluid and errors of injection are corrected by adding an IS. the exception of hydrolysis, the entire procedure is tested against distilled water. Factors which differ markedly from 1.0 indicate incorrect operations (production of incomplete derivatives, quality of the cohrmrr used, background from reagents used, including Helix ~o~at~a). This test was performed once daily. (3) Hydrolysis is checked by assaying an urine having a known steroid concentration. In addition, this urine is used to check day-to-day repeatability. (2) With

RESULTS

Evaluation of the method Hydrolysis. The mixture stored in the incubator contained 5000 Fisman units

of glucuronidase per ml and 40000 Roy units of sulphatase per ml. The effects of the temperature, hydrolysis time and enzyme concentration were studied, and hydrolysis, particulary that of dehydroepiandrosterone sulphate, was found to be incomplete when hydrolysis times were reduced. Hydrolysis was tested for completeness by extracting the hydrolysed sample with ethyl acetate and solvolysis overnight with sulfuric acid. The steroid material found was not more than with enzymatic hydrolysis alone. Formation of derivatives. From a purely analytical point of view, converting all hydroxyl groups into TMS ether groups is a highly attractive procedure as thermal stability increases, column adsorption decreases, elution times are often reduced” and more reproducible results are obtained. To prevent enol formation in the preparation of TMS ethers from hydroxyketosteroids, MO-TMS derivatives are the derivatives of choice4s5. Surprisingly, the silyIation times obtained were much shorter than those referred to in the literature4B5. The substance used as a model in silylation experiments was pregnanetriol (301,17.x, zoa). Fig. I shows that silylation of pregnanetriol is catalyzed to a high degree by metlloxamine hydrochloride. The reaction rate was measured by determining the peak ratios of di- and tri-t~methylsilyloxypregnanetriol at the times stated (methylene unit (MU) values 29.05 and 28.00 respectively). Fig. I shows that a silylation time of 6 h will suffice (75% of @cortol is converted into the pentatrimethylsilyloxy compound within 6 h). Gas chromatography

Using more selective phases such as OV-17 and OV-225 was found to be disadvantageous, particularly in separating 17-OH corticosteroids. Steel columns cannot be used because of the partial breakdown of certain x7-OH corticosteroids. Using z.5-mm columns rather than the more conventional 4-mm columns was essential to ensure adequate separation.

106

VAN DE CALSEYDE

et fd.

!

I

100

2 2 .E *

/

I

I

/

20/

/

/

fX

9’

P’

5 e z2

4 Time

6

,‘I’

24

-hours

Fig. I. Influence of adding I .5 mg methoxyamine-HCl of pregnanetriol. o-.-.0, without methoxyamine-HCl. X-.-.X, with methoxyamine-HCl.

to the silylation

mixture

on the silylation

Qzsantitative aspects

Repeatability of calibration testing within a single day was excellent (coefficient of variation 5%). Day-to-day relatability was rather less satisfactory (coefficient of variation IO%) as column conditions vary slightly from one day to another. When a column has been used for approximately two months, injecting IO profiles daily, factors will deteriorate (f > x.3) and the column has to be refilled. Controlled 24-h specimens of urine were collected from 30 normal adults (males and females). The mean and SD. were calculated for the most important steroids, and are listed in Table I. The series of observations is too limited to refer to the values as normal but the values calculated are well in accordance with the results reported by others2.

Fig. 2 shows the gas chromatogram of a calibration mixture (numbers correspond to Table I). Fig. 3 shows the relative differences in retention time (for MU values, see Table I) for the most important urinary steroid metabolites. Fig. 4 is a chromatogram of the urinary steroids of a normal adult male. Fig. 5 shows the urinary steroids of a {r4-year-old) boy with an adrenogenital syndrome (zr-hydroxylase deficiency). Typical metabolites are pregnanetriol, pregnanetriolone and r7-OH-pregnanolone. Fig. 6 shows the urinary steroid profile of a (4r-year-old) female with Cushing’s syndrome. Outstanding metabolites are HA, THE, THF+aIlo-THF, cr-cortolone and /?-cortol. Fig. 7 shows the urinary steroid profile of a (3o-year-old) female with SteinLeventhal syndrome. r7-Keto-steroids, namely dehydroepiandrosterone and andro-

.E

!S Y ,” w

y” S

W $!

ADULTS

name and abbreviation

OF NORMAL

Androsterone (A) Etiocholanolone (E) Dehydroepiandrosterone (D) I I-Ketoandrosterone (KA) II-Ketoetiocholanolone (KE) 17-OH Pregnanolone (17-OHP,) I I-OH Androsterone (HA) II-OH Etiocholanolone (HE) Pregnanediol (Pr) Pregnanetriol (P,) I x-Deoxytetrabydrocortisol (THS) Pregnanetriolone (P,one) Pregnanetetrol (PJ Pregnenetriol (P&) Tetrahydrocortisone (THE) Tetrahydrocortisol (THF) Allotetrahydrocortisol (allo-THF) Cortolone /j-Cortol Cholesterol (chol) Cortol

Trivial

STEROIDS

I

5a-androstane-3a-ol-I7-one $androstane-3a-ol-I7-one 5-androstane-3/?-ol-I7-one 5cr-androstane-3cc-01-I I, I7-dione 5/Gandrostane-3a-ol-II, x7-dione 5p-pregnane-3a, I7a-diol-zo-one 5a-androstane-3a, 1x/?-diol-I7-one 5&androstane-3a, 1Ij3-diol-I7-one 5p-pregnane-3r, zoa-diol 5B-pregnane-3rx, 17x, noa-trio1 +pregnane-3ff, 17x, zr-triol-zo-one +pregnane-ja, I7a,zoa-triol-II-one 5/3-pregnane-3a, I I/3. I7a, noa-tetrol 5-pregnene-3@, I7c(, zoa-trio1 5p-pregnane-3a, 17cc, 2I-triol-I I, 2o-dione 5p-pregnane-3a, rrfl, I7cr, zr-tetrol-zo-one 5a-pregnane-3a, II@, r7a, zr-tetrol-20.one 5@-pregnane-301, I7a, 2oa, 2I-tetrol-II-one 5/?-pregnane-3cc, IIP, I7a, 2op, zr-pent01 5-cholestene-38-01 $pregnane-3cr, II/?, I7a, 2oc(, ar-pent01

Official name

1 Numbers correspond to numbers in Figs. 2-7. z MU (methylene unit) values were determined by Chambaz and Horning’s method4. 3 These values are based on steroid analysis in 15 adult males and 15 adult females. * This value is the sum of constituents 6 and 7.

13 I4 15

12

II

10

9

;:

:

3 4

2

I

Steroid rtumherl

URINARY

TABLE

25.01 25.22 25.63 26.00 26.10 26.90 26.93 27.~2 27.58 28.00 28.66 29.02 29.25 29.43 29.60 30.23 30.34 30.51 30.79 30~79 3s.22

MU value of MO-TMS2 derivative

Mean

0.9 0.9 0.4 0.4 0.3

-

4.0 & 1.0

0.9

-

-

I__ 3.3 sr 0.9

3.3 f

-

1.1

3.8 i

I.4

1.3 & 0.6 0.4 * 0.2 0 -

0.2

0.6~ 0.3 0.6 0.6

0 -

* * f & *

3.2 i

1.0 & 0.54 1.0 “c 0.5

I.2

2.3 * I.1 0.8 & 0.4 0.6 & 0.3 -

4.0 f

1.8 I.0 I.3 1.3 0.4

rt i* * &

mk-P-4h 2.3 2.3 0.8 0.8 0.6 -

-

females

3.8 & 1.I

_

& S.D.3

v/z+ h

males

VAN DE CALSEYDE

108

I

1,: 3 5

89

I10 11

1’. II I

t

16

2’0 Time

Fig. 2. GLC separation of a calibration

12

3

45

10

678

control. (IS is the internal standard tetracosan.)

11 12131415

‘2

1617182C 2-l

I

I 1. 24

36 min

28

26

Fig. 3. Diagram of methylation

30

32

unit (MU) values. For exact values see Table I

--z----

lb-

---

Time-min

Fig. 4. Urinary steroid profile of a normal adult male. C&n. Chirn. Acta.

38

(1972)

103-111

30

et d.

PROFILING

URINARY

STEROIDS

I09

6

t

llo

20 Tome-

+ ! 30

min

5. Urinary steroid profile of a boy (aged 14 yrs.) with adrenogenital syndrome (rI-hydroxylase deficiency). Internal standard: 40 ,ug tetracosan.

Fig.

‘;:

15

3

71

7

19

5

I/ II I

!

J(

I

;-.,

Fig. 6. Steroid profile of a female (aged qr yrs.) with Cushing’s syndrome.

sterone, are elevated. 17-OH corticosteroids (THE and THF) are low but normal. Further studies are required to decide whether these changes are specific of this syndrome. DISCUSSION

Steroid profiling by GLC was introduced by Gardiner and Horning11 in 1966. The procedure proposed by us is a continuation of their excellent work in this field, CL&z. Chinz.Acta, 38 (1972) 1o3-rrr

VAN DE CALSEYDE

t__-

-. -.. -. -__+-_-_ IO

-_-

20 Time

Fig. 7. Urinary

steroid

et Cd.

30 - min

profile of a female (aged 39 yrs.) with Stein-Leventhal

syndrome.

and was found to be satisfactory after almost one year of practical experience. The modifications introduced by us have enhanced the accuracy and reliability of the assay. The most important modifications are: (a) A smaller quantity of urine, which made it economically possible to use a higher enzyme concentration and thus to reduce the effect of enzyme inhibitors, if any, in addition to ensuring shorter hydrolysis times. Moreover, using smaller portions of urine all operations can be carried out in standard centrifuge tubes. (b) Extraction with dichloromethane rather than with ether. Unlike in the procedure of Bacchusl” the use of ethyl acetate was not necessary for the extraction of the more polar compounds. (c) Complete silylation of OH groups (including the tertiary 17-OH group) using a strong silylation mixture offers the following advantages. Elution times were reduced, which made it possible to determine a profile within approximately 30 min. In addition, calibration checking was found to produce repeatable results. Although it takes two days until the results of anaIysis are known, the procedure is not laborious, so that the number of estimations performed daily by a single analyst depends only on the number of columns available. Two assays per hour can be performed per column. Because of the high degree of reproducibility, duplicate determinations were found to be unnecessary. The two-day interval before the result is known is a matter of minor importance in endocrinological diagnosis. Determining a steroid profile was to a large extent substituted for the following estimations in our laboratory: colorimetric total assay of r7-ketosteroids and ketogenic substances, separate determination of pregnanetrioP, the ketosteroid diagram” and estimation of THS, THE, and THF by column chromatography12.

PROFILING

URINARY

III

STEROIDS

REFERENCES I PERVESTERGAARD,C&. Chem., 16(197o)651. z L.P.CAWLEY,B.O.MUSSERAND H. A.TRETBAR,Amer.J.CZin.Pathol.,48(1g67) 216. 3 W. J. A. VAN DEN HEUVEL AND E. C. HORNING, Biochem. Biophys. Res. Commun., 3 (1960) 356. 4 E M. CHAMBAZ AND E. C. HORNING, Anal. Biochem., 30 (1969) 7. =, E. M. CHAMBAZ AND E. C. HORNING, Anal. Letters, I (1967) 201. 6 J. A. VBLLMIIG, Chromatographia, 3 (1970) 233. 7 J. A. VGLLMIN,Chvomatogvaphia, 3 (1970) 238. 8 C. A. CRAMERS, Symposium Gas Chromatography in Clinical Chemistry, Nijmegen, 1971. g H. GLEISPACH,2. Klin. Chem. Klin. Biochem., 7 (1969) 379. IO H. BACCHUS, C&z. Chem., 13 (1967) 855. II W. L. GARDINER AND E. C. HORNING, Biochim. Biophys. Ada, 115 (1966) 524. 12

E.W.M.G.

PIETERSE,R.J.H.SCHOLTISANDN.A.SCHMIDT.C~~~. Chim.Acta.

25(196g)

.243.

CZin. Chim. Acta, 38 (1972) 103-111